Homing is the phenomenon where transplanted hematopoietic cells find their way into the recipient's bone marrow. This article describes a protocol for studying the homing of hematopoietic cells to the relative areas in the bone marrow. By isolating the cell population of interest, staining this population with a dye and injecting them into the bloodstream of a recipient animal.
The percentage of cells positive for the dye in the animal is then determined after a period of time. With this method, the homing efficiency of these cells from two genetically distinct animals can be compared. Hi, my name is RHI Reen Yusef, and I'm a postdoctoral fellow in Dr.David Skadden's laboratory at the Center for Regenerative Medicine at Massachusetts General Hospital and the Harvard Stem Cell Institute.
Today we're going to be showing you a procedure to study the homing of hematopoietic stem cells to the bone marrow where they normally reside. We use this protocol in our laboratory to study the differences between the homing of hematopoietic stem cells from various genetically altered mice. So let's get started.
To begin the homing experiment, we extract bone marrow from both WT and lysophosphatidic receptor one or LPA R one knockout animals. All mouse dissection should be done in a laminar flow tissue culture hood, especially if the cells are going to be transplanted into a recipient animal For dissection, first place a liner on the bottom of the hood. Next place dissection tools on the liner, including a pair of autoclave, forceps and sharp pair of scissors, a sterile disposable scalpel and alcohol swaps.
Next place in the hood, a bottle of modified PBSA sterile six well plate a mortar and pestle, A 50 cc blue topped conical tube and a disposable filter. Following CO2 mediated euthanasia, sterilized the mouse's body in 70%isopropanol for five seconds to prevent contamination of the cells of interest. With mouse fur, the step does not seem to compromise the yield of cells or experimental results.
After sterilizing the mouse, use a pair of forceps and sharp scissors to make a small snip in the ventral skin of the mouse over the abdomen. Next, using your hands stretch the skin off. The skin should come off the hind limbs.
Easily extract the femur and tibia carefully to prevent dislodging the head of the femur. Since this contains a large amount of bone marrow. Next, detach the upper limbs from the animal's torso by simply applying traction to them while keeping the torso immobile.
This removes the upper limbs as one piece. Extract the hum eye from these upper limbs. Place the collected bones in modified PBS in six well plates labeled for WT samples.
Repeat this dissection for KO mice and collect in a six well plate with modified PBS this time labeled for KO samples. Once dissection is completed, dispose of the mouse carcasses and place a new liner in the hood. Then clean the bones using a disposable scalpel.
Place the cleaned bones in the mortar one mouse at a time. Today we've started with the WT samples. Add two milliliters of modified PBS to the mortar and crush the bones using a pestle.
First fragment the bones by moving the pestle back and forth 10 times. Next, pulverize the bones with circular rotary movements. You'll do a series of 50 clockwise, then 50 counterclockwise movements.
After the first set of 50 circular movements, transfer the fluid from the mortar to a 70 micrometer filter placed on top of a 50 milliliter conical tube. Do not forget to add two milliliters of PBS to the mortar. After every transfer of the filter, this will prevent the bones drying and leading to apoptosis of the cells.
Once the material in the mortar becomes white, transfer the material left in the filter back to the mortar and begin crushing again. This ensures that the cells in the filter are not wasted. Repeat a set of circular movements one or two times to make the material in the mortar whiten color, which indicates the extraction is complete.
Repeat this procedure for samples from the ka o mouse. Don't worry. The rest of the protocol is much less labor intensive.
To collect the cells centrifuge, the individual WT and KO sample solution in conical tubes at 1200 RPM for 10 minutes aspirate off the super natin using separate aspirator pipettes for each tube. Next, add two cc of sterile, a CK lysis buffer to each pellet and incubate on ice. For five minutes, add h CCC of modified PBS to each tube.
Then take 10 microliter samples from each tube and pipette into two separate wells. In a 96 well plate clearly labeled WT and kko. Place the tubes in the centrifuge and spin them at 1200 RPM for 10 minutes.
While the tubes are spinning, add 30 microliters of trian blue dye and 60 microliters of modified PBS to each well of the 96 well plate with the cells of interest. Mix the cells and die well by pipetting up and down. Remove the tubes from the centrifuge and resus.
Suspend the cells in PBS without calcium, magnesium, or L-glutamine at a concentration of 20 million cells per milliliter. This often averages to be about five milliliters of volume each for WT and KO samples. To label the cells add to each tube the cell staining dye die eye to a final concentration of five micro molars.
Vortex immediately for 10 seconds after the dye is well mixed with the samples. Incubated 37 degrees for 10 minutes and avoid exposure to light. Next, fill the tubes with regular PBS, not modified PBS and spin for 10 minutes.
At 1200 RPM, continue to use this PBS rather than the modified PBS From this point on. After centrifugation, aspirate the super natin and resuspend the cells in PBS at 40 million cells per milliliter. Load 500 microliters into one cc syringes with a 27 gauge needle.
Five syringes for the WT mice and five syringes for the K mice. Cover the syringes with foil in order to keep the dye eye from disintegrating. Inject 500 microliters of the cell mixture into each recipient animal, which has been irradiated with nine grays of irradiation four to 24 hours prior to injection.
Having an extra syringe ready can help as tail injections can be challenging. Eight hours after injection harvest the bone marrow from the hind limbs of each recipient animal. Next process, the bone marrow is done previously with donor samples.
Resus suspend the cells from each animal in one cc of PBS and count the cell population using a hemo cytometer. Finally, analyze the samples on the LSR two flow cytometer from BD or an equivalent instrument. We've just shown you how to perform a homing assay on hematopoietic stem cells.
The number of cells that you choose to inject into the recipient mice, the dye that you use to stain these cells and the time at which the recipients have sacrificed are all variables that will need to be predetermined. Before you perform your experiment, your question and hypothesis will determine the choice of these variables. So that's it.
Thank you for watching and good luck with your experiments.
